Antibodies are substances contained in the blood and other fluids of the body, as well as in the tissues, and which bind to antigen to make it innocuous. Antibodies constitute one of the natural defense mechanisms of the body. They are highly specific and they kill, bind or make innocuous the antigen which induced their formation.
The antigen in contact with the immune system, thus activates a complex series of cellular interactions to eliminate the antigen and/or to re-establish the preceding equilibrium.
Two of the characteristic features of antigens are their immunogenicity, that is their capacity to induce an immune response in vivo (including the formation of specific antibodies), and their antigenicity, that is their capacity to be selectively recognized by the antibodies whose origins are the antigens.
It is known how to stimulate the immune response deliberately by administering a specific antigen by means of a vaccine. The procedure allows the retention of a state of immune response in the organism which allows a more rapid and more effective response of the organism during subsequent contact with the antigen.
However, some antigens have only a weak immunogenicity and they induce an insufficient immune response to produce an effective protection for the organism. This immunogenicity can significantly be improved if an antigen is co-administered with an adjuvant.
Adjuvants are substances that enhance the immune response to antigens, but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants are normally the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune response.
Aluminium hydroxide and aluminium phosphate (collectively referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.
A wide range of extrinsic adjuvants can provoke immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP).
Chemically defined adjuvants, such as monophosphoryl lipid A, phospholipid conjungates have been investigated (see Goodman-Snitkoff et al., J. Immunol. 147:410-415 (1991) as has encapsulation of the protein with a proteoliposome (see Miller et al., J. Exp. Med. 176:1739-1744 (1992)).
Synthetic polymers have also been evaluated as adjuvants. These include the homo- and copolymers of lactic and glycolic acid, which have been used to produce microspheres that encapsulate antigens (see Eldridge et al., Mol. Immunol. 28:287-294 (1993)).
Nonionic block copolymers are another synthetic adjuvant being evaluated. Adjuvant effects have also been investigated for low molecular weight copolymers in oil-based emulsions (see Hunter et al., The Theory and Partical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.) John Wiley and Sons, NY. Pp. 51-94 (1995)) and for high molecular weight copolymers in aqueous formulations (Todd et al., Vaccine 15:564-570 (1997)).
Desirable characteristics of ideal adjuvants are lack of toxicity and an ability to stimulate a long lasting immune response. One of the most commonly used adjuvants in humans is alum. Other adjuvants, such as Saponin, Quil A and the water in oil adjuvant, Freund's with killed tubercle bacilli (Freund's complete) or without bacilli (Freud's incomplete), have had limited use in humans due to their toxic effects; and concerns have been raised as to undesirable effects in animals.
Simply said, many adjuvant formulations have been described but most are never accepted for routine vaccines, and few have been approved for use in humans. This is mainly due to their toxicity. For example, the mineral oils used as adjuvants in certain animal vaccines are not readily degraded and persist at the site of injection thereby causing unacceptable granulomas; and, in general adjuvant formulations such as mineral compounds oil emulsions, liposomes and biodegradable polymer microspheres cause local reactions due to depot formation at the site of injection.
Examples of adjuvants presently approved in human vaccines include Alum, MF59 (an oil in water emulsion), MPL (a glycolipid), VLR, Immunopotentiating Reconstituted Influenza Virosomes (IRIV) and cholera toxin (see Reed et al. Trends in Immunology 30:23-32 (2008).
One group of adjuvants known in the art are the so called sulpholipopolysaccharides, i.e. polysaccharides containing both fatty acid esters and sulphate esters (Hilgers et al., Immunology 60, pp. 141-146, 1986). A method for preparing these compounds has been described in the international patent application WO96/20222 and WO 96/20008. These methods for preparing sulpholipopolysaccharides result in the formation of different sulpholipopolysaccharides derivates varying in the number of fatty acids esters present per polysaccharide molecule, the number of sulphate esters present per polysaccharide molecule, the number of hydroxyl groups per polysaccharide molecule and the distribution of the fatty acid esters, the sulphate esters and the hydroxyl groups over the polysaccharide molecule. This means that during preparation of these sulpholipopolysaccharides a mixture is obtained of many different sulpholipopolysaccharides. Consequently, the yield of the desired sulpholipopolysaccharide is relatively low or the adjuvant needs to be used as a difficult to characterise mixture causing regulatory issues.
In the European patent EP 1233969 an adjuvant composition is claimed which adjuvant comprises sulpholipodisaccharides. Also a method is described for preparing these sulpholipodisaccharides. In one of the embodiments the sulpholipodisaccharides prepared are fully substituted with fatty acid ester or sulphate ester groups. However, as will be further described in the following, when these sulpholipodisaccharides are used as adjuvants in animals, undesired side effects such as occurrence of mean body temperature rise (including fever) and local irritation (tissue swelling) occur.